(1) Field of the Invention
The invention relates to a method for detecting light signals, in which a light signal impinges on an optoelectronic converter, where the light is converted into an electric signal, and in which, after the conversion, the electric signal is distributed to several analysis channels. Here, in each analysis channel (i) a signal analysis is performed, which is different from the signal analyses for the other analysis channels and (ii) an output signal is produced. The invention relates to the problems found in optic examination methods, especially in microscopy, where many different types of detection methods can be used. Each of the methods has advantages and disadvantages and some of the methods may beneficially be used only in special examination methods. For this reason, detection methods are frequently used alternately or in combinations. The invention also relates to a detector module for detecting and analyzing light signals as well as the use of such a detector module in a laser-scanning method.
(2) Description of the Related Art
In microscopy, in general, and in laser-scanning microscopy, in particular, there are a multitude of different examination methods, each requiring a detection method adjusted to the examination method. Each of these detection methods has its own characteristic and is generally particularly well suited for one or several of the examination methods, but poorly adapted for other methods.
A widely used method is the integration of signals within a defined measurement period with a subsequent analogue-digital conversion, for example. Usually capacitors are used for the integration and collect a charge in a specified measurement period. Light signals are converted into electric signals by an optoelectronic converter, so that the charge collected in the measurement period corresponds to the light intensity. Between two integration processes, the capacitor must be discharged and/or removed, so that a certain downtime develops, in which no integration can occur. Up to 30% of the overall time is required to discharge the integration capacitor, in which time no signal can be detected; as a consequence loss of sensitivity develops. In this case the so-called odd/even variant is often used as a solution. Here, while one capacitor is prepared for the next integration process, i.e. its charge is being removed, another capacitor is used for integration. This integration method has a very wide dynamic range when the integration time constant is adjusted appropriately. The loss in sensitivity can be avoided in the odd/even variant; however, in this method lines develop in the image, which result from the tolerances of the two integrators and their parts during switching.
Another method is to count individual photons. This method is very sensitive; however it only has a narrow dynamic range and is therefore only operational to a limited extent. The same applies to the so-called 2D-photon counting, such as for example described in US 2003/0183754 A1. When a photon multiplier, for example a photomultiplier-tube (PMT), is used as a detector, the highest possible voltage must be connected for detecting individual photons in order to receive a signal. When several photons simultaneously impinge the detector, they have no influence on the intensity of the signal, because the signal is already at maximum level with a single photon. There is therefore no difference between the impingement of few or many photons; the dynamics are therefore very low.
Furthermore, a method that can be easily implemented is the so-called oversampling method. This method is particularly well suited to scan a changing signal because the signal is scanned at a higher scanning frequency than actually necessary to represent the band of the signal. This way, during the measurement period the signal-to-noise ratio can be influenced.
In addition to these standard methods in laser scanning microscopy, additional detection methods are used specially adjusted to the examination methods. In so-called Fluorescence Lifetime Imaging Microscopy (FLIM), a pulsed illumination system is necessary and very fast with digital signal processing in the pico second range. The pulse time, i.e. the time at which the molecules are excited to fluorescence, plays an important role. For this reason, processing of the signals usually occurs in different steps: for example the preprocessing is frequently performed in the detection module, while the final processing may occur in a computer for example. In order to determine the life of a fluorescence excitation, the time between the excitation and the detection of the signal must be determined.
A similar, special detection method is used for the fluorescence-correlation spectroscopic measurements (FCS-measurements). Here, bonding features of molecules are determined within the cofocal volume detected by a laser-scanning microscope. For this purpose, the emission signals of fluorescent molecules must be detected; here too the temporal progression and interval the signals are detected are of decisive importance.
For simultaneous or alternating use of several detection methods, the prior art suggests different solutions. In US 2006/0203241 A1, a device for the spectral selection and detection of spectral ranges of a light beam are disclosed, in which the light beam is split into several, even spectrally different, partial beams. Each of the partial beams impinges a different detector, with the detectors each having different detection features and/or different detection methods. This construction is very expensive because for each partial beam a separate detector with a separate optoelectronic converter is used.
From an article by W. Becker et al., “Proceedings of SPIE,” vol. 4431, pages 94-98, a method for detecting an object with the help of a laser scanning microscope is known. The detection device includes two detection channels, with prior to the detection the signal first being split optically into two channels. Each of the channels has therefore a separate optoelectronic converter. A separate analysis device is connected to each converter.
In contrast thereto, the solution disclosed in DE 102 53 108 B4 shows an improvement. Here, the light is detected by a single detector, the detection signal is then distributed to two channels via highpass filters and/or lowpass filters. According to the disclosure of DE 102 53 108 B4, these filters are necessary to achieve a clean channel separation. In one of the two channels pulses are created by a pulse former, which serves for further processing. The detection signal originally connected to the input of the second channel no longer requires any attention, so that the second channel essentially acts as a pulse counter, which can also register the temporal intervals between the pulses. The system presented in DE 102 53 108 B4 is a very special detector, which can only be used for certain problem conditions, such as for example FCS-measurements.
The object of the invention is therefore to develop a method and an arrangement which has as wide a range of application as possible and can flexibly be adjusted to different examination conditions, in particular in microscopy and laser scanning microscopy.